Isolation, identification, and characterization of an aluminum-tolerant bacterium Burkholderia sp. SB1 from an acidic red soil

Aluminum (Al) toxicity usually occurs in acidic soils worldwide, which is detrimental to the growth of organisms. An Al-tolerant bacterium, SB1, was isolated from an acidic red soil of Chingkang Mountain, located in Jiangxi Province of China. Polyphasic analysis, including a 16S rDNA phylogenetic tree as well as morphological and physicochemical properties, revealed that the isolate was a gramnegative, rod-shaped bacterium, which was recognized as Burkholderia sp. SB1 and had extreme acidity tolerance (pH 2.2) and excellent Al resistance (270 mg L^-1 Al³⁺). It could remove Al by up to 97.7% at a concentration of 54 mg L^-1 Al³⁺. SB1 behavior under different temperatures and antibiotics was also examined. SB1 preferred moderate temperature conditions, ranging from 25 to 37℃, and exhibited notable resistance to multiple antibiotics (including ampicillin, streptomycin, and tetracycline), except for being sensitive to chloramphenicol. Therefore, as the first reported bacterium to possess favorable Al resistance and excellent Al removal, Burkholderia sp. SB1 can potentially be used as an agent for bioremediation of Al-contaminated acidic red soils.     

Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.     

Isolation and biochemical characterization of an antifungal peptide from Amaranthus hypochondriacus seeds

An antifungal peptide, Ay-AMP, was isolated from Amaranthus hypochondriacus seeds by acidic extraction and then purified by reverse-phase high-pressure liquid chromatography. The molecular mass of this peptide, as determined by mass spectrometry, is 3184 Da. The peptide belongs to the superfamily of chitin-binding proteins, containing a single cysteine/glycine-rich chitin-binding domain, and it was found that Ay-AMP degrades chitin. Ay-AMP inhibits the growth, at very low doses, of different pathogenic fungi, such as Candida albicans, Trichoderma sp., Fusarium solani, Penicillium chrysogenum, Geotrichum candidum, Aspergillus candidus, Aspergillus schraceus, and Alternaria alternata. Ay-AMP is very resistant to the effect of proteases and heating; however, it showed an antagonistic effect with CaCl2 and KCl.     

酸性土壤中解磷菌的分离及其促生效果研究

酸性土壤中磷易被固定,磷的生物有效性极低。解磷菌对土壤中难溶性磷具有重要的增溶作用。虽然已有不少解磷菌方面的研究,但是主要集中于中性和石灰性土壤中钙磷的解磷菌报道,而关于酸性土壤中高效溶解铝磷的微生物报道较少。采用培养基和土培试验,首先对酸性土壤上不同植物（胡枝子、大豆、水稻）根际土壤中的解磷菌进行了分离,然后比较了它们对不同磷源（磷酸钙和磷酸铝）的溶解能力,最后研究了它们对大豆生长和磷吸收的影响。通过使用难溶性磷源（磷酸钙和磷酸铝）的固体培养基,分离得到5株优势菌株L1、S1、S2、R1和R2,经16S rRNA序列鉴定,L1属于阮杆菌属（Nguyenibacter）,S1和S2分别属于假单胞菌属（Pseudomonas）和沙雷氏菌属（Serratia）,R1和R2分别属于伯克氏菌属（Burkholderia）和雷尔氏菌属（Ralstonia）。菌株S1、S2、R1和R2对难溶性磷酸钙有较强的溶解能力,对磷酸铝的溶解能力较弱;菌株L1对磷酸铝表现出较高的溶解能力,对难溶性磷酸钙的溶解能力弱。联合接种菌株L1+S1对大豆生长和磷吸收表现出良好的促进效果,而单独接种L1和S1效果不显著。...     

Isolation and characterization of immunostimulatory polysaccharide from an herb tea, Gynostemma pentaphyllum Makino

Water-soluble polysaccharide from Gynostemma pentaphyllum herb tea (PSGP) was isolated by hot-water extraction and ethanol precipitation. The chemical components and preliminary immunomodulating activity of PSGP were investigated both in vitro and in vivo. Capillary zone electrophoresis analysis showed that PSGP was a typical nonstarch heteropolysaccharide, with glucose being the main component monosaccharide (23.2%), followed by galactose (18.9%), arabinose (10.5%), rhamnose (7.7%), galacturonic acid (4.7%), xylose (3.9%), mannose (3.1%), and glucuronic acid (1.2%). PSGP could significantly stimulate peritoneal macrophages to release nitric oxide, reactive oxygen species, and tumor necrosis factor-alpha in a dose-dependent manner. This immunostimulating activity of PSGP was further demonstrated by its inhibition on the proliferation of human colon carcinoma HT-29 and SW-116 cells incubated with the supernatant of PSGP-stimulated macrophage culture. It is evident that PSGP is a very important ingredient responsible for at least in part the immunomodulating activity of G. pentaphyllum herb tea.     

Isolation and characterization of superoxide dismutase from wheat seedlings

Two major superoxide dismutases (SODs; SODs I and II) were found in the crude enzyme extract of wheat seedlings after heat treatment, ammonium sulfate fractionation, anionic exchange chromatography, and gel permeation chromatography. The purification fold for SODs I and II were 154 and 98, and the yields were 11 and 2.4%, respectively. SOD I was further characterized. It was found that SOD I from wheat seedlings is a homodimer, with a subunit molecular mass of 23 kDa. Isoelectric focusing electrophoresis (IEF) and zymogram staining results indicated that the isoelectric point of SOD I is 3.95. It belongs to the MnSOD category due to the fact that it was insensitive to KCN or hydrogen peroxide inhibitor. This MnSOD from wheat seedlings was found to be stable over pH 7-9, with an optimum pH of 8, but was sensitive to extreme pH, particularly to acidic pH. It was stable over a wide range of temperatures (5-50 degrees C). Thermal inactivation of wheat seedling MnSOD followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy for thermal inactivation of wheat seedling MnSOD in the temperature range of 50-70 degrees C was found to be 150 kJ/mol. HgCl2 and SDS at a concentration of 1.0 mM significantly inhibited enzyme activity. Chemical modification agents, including diethyl pyrocarbonate (2.5 mM) and Woodward's reagent K (50 mM), significantly inhibited the activity of wheat seedling SOD, implying that imidazole groups from histidine and carboxyl groups from aspartic acid and glutamic acid are probably located at or near the active site of the enzyme.     

Isolation and characterization of two endoxylanases from Fusarium graminearum

This paper reports the first isolation from cultures of two endoxylanases secreted by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch]. When F. graminearum is grown on wheat bran hydrated with a modified synthetic medium, high xylanase activity can be extracted. The two endoxylanases were identified by LC-MS/MS as the products of genes FGSG_6445 (Genbank gene id 2788192 ) (xylanase 1) and FGSG_3624 (GenBank accession no. AJ863566 ) (xylanase 2) with 61 and 51% sequence coverage, respectively. Both enzymes showed a pH optimum at pH 6, with xylanase 1 exhibiting a wider active pH range (5.5-9) than xlylanase 2 (5.5-7.5). Their temperature dependences were similar, >60% between 35 and 60 °C, with optimal temperatures of 45 °C for xylanase 1 and 50 °C for xylanase 2. Kinetic studies found that both enzymes had a lower K(m) for linear beachwood xylan than arabinoxylan. For xylanase 2, the V(max) increased with arabinoxylan, but decreased for xylanase 1.     

Isolation and characterization of novel stilbene derivatives from Riesling wine

Besides the already known stilbenes trans-resveratrol as well as isomeric piceids seven novel stilbene derivatives have been isolated from a commercial Riesling wine. The newly identified compounds included the monostilbene 2,4,6-trihydroxyphenanthrene-2-O-glucoside, as well as two isomeric resveratrol-2-C-glucosides. In addition, four dimeric stilbenes, i.e., cis- and trans-epsilon-viniferin diglucoside as well as pallidol glucoside and pallidol diglucoside, have also been obtained for the first time from Riesling wine.     

Isolation and characterization of endothelium-dependent vasorelaxing compounds from grape seeds

Previous work has shown that red wines, grape juices, and other grape products cause endothelium-dependent relaxation (EDR) of blood vessels in vitro by increasing nitric oxide production. In this paper we describe the isolation and characterization of some of the compounds responsible for EDR activity. Concord grape seeds were extracted with methanol and the compounds were separated by Toyopearl TSK HW-40S chromatography. Resulting fractions (primarily phenolic acids, catechins, and proanthocyanidins) were further separated semipreparatively by reversed-phase HPLC, and peaks were collected and bioassayed for EDR activity using the rat aorta preparation. EDR-active compounds were subsequently characterized by HPLC retention times and electrospray-ion-trap mass spectrometry. The compounds exhibiting the most EDR activity were proanthocyanidin trimers, tetramers, pentamers, and polymers and their gallates, as well as a dimer gallate (EC50 values in the range of 0.6-2.5 microg catechin equivalents/mL). These compounds should be useful for in vitro and in vivo studies, particularly as they relate to improvement of cardiovascular function.     

Isolation and characterization of Thiobacillus from alkali soils

Alkali soils are poor in sulphur-oxidizing bacteria. By appropriate enrichment, two bacterial strains resembling Thiobacillus thiooxidans and T. novellus have been isolated from an alkali soil. The strain of T. novellus possesses characteristics which make it suitable in reclamation of alkali soils by sulphur amendment.     

Hydroxyl release by maize (Zea mays L.) roots under acidic conditions due to nitrate absorption and its potential to ameliorate an acidic Ultisol

Purpose

Hydroxyl ion release by maize (Zea mays L.) roots under acidic conditions was investigated with a view to develop a bioremediation method for ameliorating acid soils in tropical and subtropical regions.

Materials and methods

Two hydroponic culture experiments and one pot experiment were conducted: pH, nitrogen state, and rhizobox condition, which investigated the effects of different nitrogen forms on hydroxyl release by maize roots under acidic conditions.

Results and discussion

The pH of the culture solution increased as culture time rose. The gradient of change increased with rising NO₃ ^?/NH₄ ⁺ molar ratios. Maize roots released more hydroxyl ions at pH 4.0 than at pH 5.0. The amount of hydroxyl ions released by maize roots at a constant pH was greater than those at a nonconstant pH. Application of calcium nitrate reduced exchangeable acidity and increased the pH in an Ultisol rhizosphere, compared with bulk soil. The increasing magnitude of soil pH was greater at higher doses of N. The absorption of NO₃ ^?–N increased as the NO₃ ^?/NH₄ ⁺ molar ratios rose, which was responsible for hydroxyl ion release and pH increases in culture solutions and rhizosphere.

Conclusions

Root-induced alkalization in the rhizosphere resulting from nitrate absorption by maize plants can be used to ameliorate acidic Ultisols.     

Base neutralizing capacity of sediments from an acidic lake

The base neutralizing capacity (BNC), or alkalinity consumption, of acidic lake sediments may influence the amount of neutralizing agent required to neutralize a lake if the sediment BNC is large relative to the BNC of overlying waters. The extent ofin situ sediment BNC in acidic Bowland Lake (pH 5.0) was inferred by (1) measuring the loss of Ca-45 to acidic sediments from labeled lake water neutralized with CaCO₃, and (2) measuring exchangeable Ca in sediments collected prior to and following neutralization of Bowland Lake with calcite (CaCO₃). The sediment BNC derived from the Ca-45 radiolabeling experiment was 0.01 mg CaCO₃ g^?1 w wt. The mean losses of Ca-45 from the aqueous phase of neutralized and untreated sediment/water mixtures were not significantly different. The mean pH of both neutralized and untreated mixtures decreased to 4.0 during the incubation, possibly because of oxidation of reduced sediments. Sediment BNC estimates derived from literature data for several lakes may be overestimated because of the inclusion of anoxic sediments containing significant amounts of reduced Fe. There was no significant difference in exchangeable Ca between sediments from untreated Bowland Lake and sediments collected 10 m after whole-lake neutralization indicating that little of the supplied alkalinity had been lost to the sediments. Hence,in situ sediment BNC was probably small in Bowland Lake.     

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.     

Responses of perch,Perca fluviatilis L., from an acidic and a neutral lake to acidic later

Responses to low pH of perch, Perca fluviatilis, from a naturally acid and a neutral lake were compared by 24 hr exposures to pH 4.6, 4.1 and 3.8 and by 72 hr exposures to pH 4.5. Plasma osmolality and plasma concentrations of Na and chloride decreased in fish from both lakes during acid exposures. Significant differences between the populations were observed at pH 4.1 and 4.5. Hematocrits of the fish from the acid lake increased rapidly and at higher pH compared with those of fish from the neutral lake. This was interpreted as an adaptation to their normal acidic environment, connected with the maintenance of red cell oxygen affinity. The perch from the acid lake maintained their muscle water balance at lower pH better than did the fish from the neutral lake.     

Isolation and characterization of a novel tannase from a metagenomic library

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS-PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca(2+), Mg(2+) and Cd(2+). In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.     

Isolation and characterization of cellulose obtained from ultrasonic irradiated sugarcane bagasse

Cell walls of sugarcane bagasse were first delignified with chlorite followed by ultrasonic irradiation and then by two-step sequential extractions at 23 degrees C with 15 and 18% KOH for 2 h, 15 and 18% NaOH for 2 h, 8 and 10% KOH for 12 h, and 8 and 10% NaOH for 12 h and by a single one-stage isolation with 10% KOH for 16 h and with 10% NaOH for 16 h, which released 79.4, 81.8, 83.6, 85.7, 61.5, and 65.6% of the original hemicelluloses, and subsequently yielded 50.7, 49.5, 48.6, 47.8, 57.2, and 55.4% of the cellulose, respectively. The six cellulosic preparations were free of bound lignin and had a purity of 77.1-90.1% with the intrinsic viscosity (eta), viscosity average degree of polymerization, and molecular weight (M(w)) ranging from 534.1 to 631.6 mL g(-1), from 1858.1 to 2238.2 mL g(-1), and from 301000 to 362600 g mol(-1), respectively. The structural features of the isolated six cellulosic samples were comparatively examined by Fourier transform infrared and cross-polarization/magic angle spinning (13)C NMR spectroscopy and X-ray diffraction, and their thermal stability was investigated by using thermogravimetric analysis. It was found that all of the cellulosic preparations have the typical cellulose I structure but the crystallinity of the SCB cellulose was lower than that of flax, cotton, and kenaf.     

Isolation and characterization of iron-reducing nitrogen-fixing saccharolytic clostridia from gley soils

Thirty strains of saccharolytic, N₂-fixing clostridia isolated from gleyed soils, capable of reducing Fe₂O₃-powder, were characterized physiologically and classified by conventional and numerical methods. After redefining the distinguishing features of saccharolytic Clostridium-species, 23 of the 30 isolates were assigned to the newly proposed species C. butyricum (12), C. saccharobutyricum (8), C. acetobutylicum (1). C. beijerinckii (1), and C. tyrobutyricum (1). Seven strains could not be classified and were regarded as intermediate forms. Numerical analysis confirmed the close relationship between 27 of the 30 isolates as well as to the type cultures used in comparison.     

Isolation and characterization of antioxidant compounds from Aspergillus candidus broth filtrate

The objectives of this study were to isolate the antioxidative components in the broth filtrate of Aspergillus candidus (CCRC 31543), to characterize their antioxidative properties, and to evaluate their safety. Three major compounds were isolated and identified as 3,3' '-dihydroxyterphenyllin, 3-hydroxyterphenyllin, and candidusin B. In the linoleic acid peroxidation system, the inhibition of peroxidation in these three compounds was greater than 95% and was significantly higher than that of alpha-tocopherol but equal to that of BHA at 12.5-200 microg/mL. As measured using the Rancimat method in lard, 3,3' '-di-OH-terphenyllin exhibited a protection factor value of 7.82, which was substantially higher than those of BHA (5.58) and alpha-tocopherol (4.29) at 200 microg/mL. 3,3' '-di-OH-terphenyllin and 3-OH-terphenyllin also exhibited marked scavenging effects on the alpha,alpha-diphenyl-beta-picrylhydrazyl radicals (94.7 and 96.0%, respectively), which were similar to those of BHA and alpha-tocopherol. Safety studies showed that these three compounds were neither cyto- nor geno-toxic toward human intestine 407 (INT 407) cells, nor mutagenic toward Salmonella typhimurium TA98 and TA100.